Electrophoresis, in general, is the phenomenon of the migration of charged particles or ions in a liquid carrier medium under the influence of an electric field. This phenomenon can be used to separate small particles which, by reasons of different surface chemical properties, exhibit different concentrations of surface charge in the given medium. Under the influence of the electrical field, the electrophoretic mobilities of the various classes of charged particles in the carrier medium will be different. A sample introduced at some point into the sheet of liquid carrier medium (buffer) diffuses slowly in a narrow band in the absence of a potential gradient; however, when the potential gradient is applied to the sheet of buffer, the sample particles are separated under the influence of the electrical field into various particle groups or components depending upon the electrophoretic mobility of the respective particles, the strength of the field, and the length of time that the particles remain in the field. Particles of similar mobility are concentrated in distinctive zones or bands at defined distances from the point of sample introduction (origin).
Blotting or transfer of electrophoretically resolved material, such as DNA, RNA, and protein, has become a standard procedure when sensitive and specific detection of biologically interesting macromolecules is required.
Electroblotting offers significant advantages over capillary blotting in that the electroblotting procedure is much quicker. Capillary transfer and electroblotting both require that the gel be placed in contact with the paper or other membrane to which the proteins or nucleic acids or other materials will be transferred. The difference between the methods is the transfer driving force. In capillary transfer the driving force is the absorptive potential of the filter paper, or other material. The transfer material, e.g., nitrocellulose or nylon, is placed between the gel and the absorptive paper. In electroblotting, however, as currently practiced the gel and transfer material are vertically suspended in a buffer tank between two electrodes. The protein or nucleic acids are thus driven out of the gel onto the transfer material using electrical potential. For example, a typical system involves placing a nylon membrane against a gelatin sheet, submerging the gel-nylon assembly vertically into a buffer solution, then applying an electric potential transversely across the assembly using the buffer solution as the conducting medium. This system typically uses two platinum wire electrodes, one on each side of a gel-nylon combination, and establishes a voltage gradient in the buffer solution. The electrodes are laid out in grid fashion and spaced at a distance from the gel and nylon to obtain a reasonably uniform electric field using the least amount of platinum.
The blotting procedure offers significant advantages. Firstly, molecules in the matrix of a gel are relatively inaccessible to probes such as antibodies. Transfer to the surface of a membrane allows analyses that are difficult or impossible in the gel. Also, since the transferred molecules are located at or near the surface of the membrane, analysis time is substantially reduced. In addition, the membranes are relatively strong and easy to handle in contrast to the gels which are easily torn. Moreover, the transferred molecules are bound to the membrane so that there is no loss of resolution while biological activity is usually retained. Thus, storage of the membrane prior to use is usually feasible.
However, electroblotting as currently practiced suffers from significant disadvantages. The electric field at the gel and nylon surfaces tends to be non-uniform and there tends to be voltage leakage around the gel-nylon assembly. The oxygen and hydrogen bubbles which are generated at the electrodes tend to impede electric current flow through the buffer solution if the electroblot is conducted in a horizontal manner. The physical supports required for the vertically suspended gel further reduce electric current flow and contribute to non-uniformity of field. The cost of platinum for the electrodes precludes its optimum usage and adds significantly to the overall cost of the equipment. A particularly troublesome disadvantage of this procedure is that it is inherently slow. Four hours of running time at 80 volts is typical.
Therefore, it is a principal object of the present invention to provide a rapid and efficient method for electroblotting.
It is a further object of the present invention to provide a method as aforesaid which eliminates the necessity for platinum electrodes.
It is an additional object of the present invention to provide a method as aforesaid which obtains high resolution and absence of diffusion.
Further objects and advantages of the present invention will appear hereinbelow.